1. General
This specification contains nucleotide and amino acid sequence information prepared using Patent In Version 3.1, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, <213> etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term “SEQ ID NO:”, followed by the sequence identifier (e.g. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>1).
The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue. M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
As used herein, the term “abnormality of glucose metabolism” shall be taken to mean one or more conditions selected from the group consisting of hyperglycemia, glucose intolerance, insulin resistance, hyperinsulinemia and β-islet cell dysfunction.
The term “elevated circulating lipid levels” shall be taken to mean a level of lipid clinically associated with an actual or enhanced risk of islet cell dysfunction or increased tendency to cell death. By “islet cell dysfunction” is meant an impaired ability of the islet cell to secrete insulin e.g., in response to glucose. Accordingly, a level of circulating lipid or amount of lipid in β-islet cells is an amount of lipid sufficient to enhance the risk of islet cell dysfunction or capable of causing actual islet cell dysfunction in a subject.
As used herein, the term “protein kinase C epsilon” or “PKCε” means an enzyme having the known substrate specificity and cofactor requirements of PKCε, and preferably, comprising an amino acid sequence that is at least about 80% identical to a sequence set forth herein as SEQ ID Nos: 2 or 4 or a portion thereof having PKCε activity. For the purposes of nomenclature, the amino acid sequences of the murine and human PKCε polypeptides are exemplified herein, as SEQ ID Nos: 2 and 4, respectively. Preferably, the percentage identity to SEQ ID NO: 2 or 4 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%. The term “PKCε” shall further be taken to mean a protein that exhibits the known biological activity of PKCε, or the known substrate and cofactor specificity of PKCε e.g., by transfer of phosphate to a substrate peptide comprising the amino acid sequence ERMRPRKRQGSVRRRV (SEQ ID NO: 5) in a calcium-independent manner and/or in response to phorbol ester.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
The embodiments of the invention described herein with respect to any single embodiment shall be taken to apply mutatis mutandis to any other embodiment of the invention described herein. In particular, the processes described herein with respect to the treatment of insulin resistance and/or the determination of modulators for the treatment of insulin resistance shall be taken to apply mutatis mutandis to processes for the treatment of glucose intolerance, hyperinsulinemia, and hyperglycaemia and/or to methods for the determination of modulatory compounds for the treatment of such conditions, particularly in obese subjects or subjects on a high-fat diet or showing elevated circulating lipid levels of having a propensity to accumulate lipid in β-islet cells or subjects suffering from NIDDM.
The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated herein by reference:    Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III;    DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;    Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, pp 1-22; Atkinson et al, pp 35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151;    Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;    Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000), ISBN 0199637970, whole of text;    Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text;    Perbal, B., A Practical Guide to Molecular Cloning (1984);    Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series;    J. F. Ramalho Ortigão, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany);    Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342    Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154.    Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.    Wünsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Müler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart    Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg.    Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg.    Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.    Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications).
2. Description of the Related Art
Noninsulin-dependent diabetes mellitus (NIDDM or Type II diabetes) is a serious health concern, particularly in more developed societies that ingest foodstuffs high in sugars and/or fats. The disease is associated with blindness, heart disease, stroke, kidney disease, hearing loss, gangrene and impotence. Type II diabetes and its complications are leading causes of premature death in the Western world.
Generally, NIDDM adversely affects the way the body converts ingested sugars and starches into glucose. In muscle, adipose (fat) and connective tissues, insulin facilitates the entry of glucose into the cells by an action on the cell membranes. In the liver, the ingested glucose is normally converted to carbon dioxide and water (50%), glycogen (5%), and fat (30-40%). The fat is stored as fat deposits. Fatty acids from the adipose tissues are circulated, returned to the liver for re-synthesis of triacylglycerol and metabolized to ketone bodies for utilization by the peripheral tissues. The fatty acids are also metabolized by other organs.
NIDDM can be viewed as a failure of pancreatic β-cells to secrete sufficient insulin to overcome insulin resistance at the level of liver and skeletal muscle (De Fronzo Diabetes 37, 667-687, 1987; Polonsky et al, N. Engl. J. Med. 334, 777-783, 1996). Although the functional defects obviously differ, there is increasing evidence that an inappropriate accumulation of lipid in each of these tissues, as a result of either oversupply or altered cellular metabolism, might be a common etiological factor in the progression of the disease (Boden et al., Proc. Assoc. Am. Phys. 111, 241-248, 1999; McGarry Diabetes 51, 7-18, 2002; Bergman et al. Trends Endocrinol Metab. 11, 351-356, 2000; Lewis et al, Endocr, Rev. 23, 201-209, 2002). In most NIDDM subjects, the metabolic entry of glucose into various “peripheral” tissues is reduced and there is increased liberation of glucose into the circulation from the liver. Thus, there is an excess of extracellular glucose and a deficiency of intracellular glucose. Elevated blood lipids and lipoproteins are a further common complication of diabetes. The cumulative effect of these diabetes-associated abnormalities is severe damage to blood vessels and nerves. Although the pancreas retains the ability to produce insulin, and in fact may produce higher man normal amounts of insulin (hyperinsulinemia), in diabetic subjects this insulin is insufficient to overcome the cellular resistance to insulin that occurs in obese subjects (i.e. “insulin resistance.
Insulin resistance can be defined as a state in which a normal amount of insulin produces a suboptimal metabolic response compared to the metabolic response of a normal or healthy subject. Insulin resistance is therefore a failure of target tissues to increase whole body glucose disposal in response to insulin. In insulin-treated patients suffering from Type II diabetes, insulin resistance is considered to be present whenever the therapeutic dose of insulin exceeds the rate of secretion of insulin of a normal or healthy subject.
Insulin resistance is commonly observed in obese subjects. It is a major determinant of Type 2 diabetes which occurs in those subjects whose β-cells fail to compensate for insulin resistance by enhanced insulin secretion.
Insulin resistance is also associated with hyperglycemia (i.e. the subject has an elevated level of blood glucose associated with elevated levels of plasma insulin), or glucose intolerance. Those skilled in the art are aware that the term “glucose intolerance” refers to a pathological state in which there is a reduced ability to metabolise glucose, as determined by a low fasting plasma glucose level (e.g., less than about 140 mg per deciliter for a human subject) and a sustained elevated plasma glucose level in a standard glucose tolerance test. For most glucose intolerant human subjects, the plasma glucose concentration following a glucose tolerance test would generally exceed about 200 mg per deciliter for a period of at least about 30 minutes or at least about 60 minutes or at least about 90 minutes following ingestion of an amount of glucose in a standard glucose tolerance test. Glucose intolerance is seen frequently in NIDDM but also occurs with other diseases and during pregnancy. Given the role of insulin in promoting the metabolism of glucose, glucose intolerance is an end-result of insulin resistance in an NIDDM subject.
Aberrant activities of protein kinase C (PKC) isoenzymes the liver and skeletal muscle (the major regulators of glucose disposal) have been correlated to insulin resistance in humans and animal models. The PKC family consists of at least 11 isoforms, grouped into the classical PKCs (PKCα, PKCβI, PKCβII, PKCγ), novel PKCs (PKCδ, PKCε, PKCη, PKCθ, PKCμ), and atypical PKCs (PKCζ, PKCκ/λ), which exhibit different substrate and cofactor requirements, and differences in their tissue localization.
Intracellular lipid accumulation has also implicated in β-islet cell dysfunction, in particular loss of secretory responsiveness to glucose, and reduced β-islet cell mass due to apoptosis.
Notwithstanding the correlations between PKC activity and lipid-induced insulin resistance, the specific PKC isoenzyme(s) involved in causing insulin resistance or glucose intolerance, and the tissue-specificity of any PKC in producing such effects in the peripheral tissues, are not known. The precise mechanisms of glucose intolerance or insulin resistance remain to be elucidated for effective and highly-specific treatment regimes to be developed.
There remains a need for effective treatments of insulin resistance and/or glucose intolerance and/or hyperglycaemia, particularly in NIDDM subjects.